Natural Killer (NK) cells have been implicated in defense against cancer, viral infection, and regulation of hemopoiesis. The long term objectives of this grant are to investigate the differentiation of NK cells by isolating progenitor cells from the adult bone marrow and placing them in culture conditions that favor the production of mature, Ly49 receptor expressing NK cells. We have developed a novel culture system in which Flt3+, Lin-, c-kit+ cells sorted from bone marrow give rise to Ly49+ NK cells in vitro. Attempts will be made to isolate NK or NK/T progenitor from these multipotent progenitors on the basis of two markers IL-7Ralpha or FcgammaRIII. NK progenitors maybe IL-7Ralpha- and NK or NK/T progenitors may be FcgammaRIII+. A common NK/T progenitor cell will also be sought in bone marrow on the basis of CD3epsilon gene expression. Current assay systems for NK development are based on bulk cultures. These will be refined to set up a clonal system in which expression of lineage specific markers and Ly49 gene transcription will be ascertained by RT-PCR. Flt3+ NK progenitors differentiate into IL-15 responsive NK precursors in vitro. These cells are NK1.1-, IL2/15Rbeta+. We will determine if such cells exist in vivo, and then determine whether these precursors are restricted solely to the NK lineage. Attempts will be made to define subsets of IL-2/15Rbeta+ NK precursors that are committed to, or highly enriched, for NK cell production. The hypothesis that MHC class I molecules influence the Ly49 receptor repertoire will be tested by the culture of NK precursor cells of defined MHC types on stroma that expresses similar or different MHC molecules. NK cells from mice transgenic for Ly49I and Ly49D will be used to determine how expression of Ly49 influences the repertoire of clonal population of NK cells. The role of signaling through Ly49 receptors in the generation of Ly49 repertoire will be investigated by use of Ly49I or Ly49D transgenic mice in which signaling through the Ly49 receptors is altered by changes in their cytoplasmic domain. Finally, we plan to set up a novel method for studying NK cell differentiation by culture of embryonic stem (ES) cells. In future studies genetic manipulations of ES cells will enable us to use this culture system to study the role of several genes in NK development. Overall these experiments will not only provide basic biologic information regarding NK cells but also allow manipulation for cancer immunotherapy.